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Direct Compressibility Measurements of up to 7 Kilobar Using Generator with Integrated Piston Position Sensor. Alexander Lazarev*, Vera Gross, Hembly Rivas, James Behnke, Edmund Y. Ting Pressure BioSciences, Inc. 14 Norfolk Ave., South Easton, MA 02375, USA

Abstract High pressure in the range of hundred to thousand bars is now recognized to have fundamental effects on the structure and function of biological systems from proteins, cell, to whole organisms. Much of the high pressure equipment in use for biological applications, however, continues to be a domain for do-it-yourself enthusiasts. As a result, basic research and understanding of mechanisms governing high-pressure unfolding of proteins, pathogen inactivation, membrane fluidity, protein dynamics and other significant effects of pressure is progressing at a limited pace; the successful industrial applications of pressure in food and biopharmaceutical industry to date remain driven by empirical optimization efforts that are lengthy and costly. Commercialization of the HUB-series pressure generators opened up new opportunities in high pressure research, particularly in studies of protein dynamics and high pressure enzymology. Modular instrument design enables fast re-configuration of the instrument to drive pressure vessels [1], optical or magnetic resonance cells [2 - 4]. Moreover, a recent addition of the piston position sensor to the HUB880 pressure generator offers direct measurements of the sample compressibility () and monitoring of transitions at pressure up to 7 kilobar using this instrument platform without a need for additional analytical equipment. This presentation will describe the principle behind direct compressibility measurements on a HUB880 platform and show examples of bulk modulus determination for several liquids. The rapid and precise bulk modulus measurements has sufficient resolution to determine the water content in extra virgin olive oil, predict flow rate fluctuations in gradient UPLC applications and potentially better understand pathogen inactivation using High Pressure Processing.

HUB880 Pressure Generator: Flow Diagram and Specifications Automatic Data Collection

MS Windows 8.1 Pressurized Pressure Pressure Tablet PC water source Transducer vessel The HUB880 was configured to output pressure from 0 to 80,000 psi (5.516 kilobar) following a slow triangular waveform to allow for dissipation of adiabatic into the metal components of the system. Isentropic experiments can be conducted using rapid pressure cycling.

Pressure Piston position sensor data were Manual recorded automatically by a control Manual or automatic override valves system along with pressure and Controls Inlet Displacement values. Data were stored as MS Windows ® .CSV Check Valve Outlet (comma-delimited file) format and Power 30 KPSI Pressure analyzed in MS Excel. Air Pressure Display HP Check Valve HP “T” Fitting Pressure InletPressure Source Regulator Error Error Sensor indicator (Air) Power Shift valve 0 indicator 160 PSI MAX

Shift Valve External Control System Isothermal Compressibility and Bulk Modulus of Samples Computer USB 2.0 Intensifier Control Extend Intensifier 1:880 The pressure dependence of is described to first order by the isothermal compressibility Command Pressure coefficient κ defined as indicator Display

HUB880 Intensifier Ratio 880:1 where V is the volume and κ is bulk modulus at constant temperature T.

Maximum Pressure 7 kbar (100,000psi)

Pressure Transducer Span 0-7 kbar Isotherms shown here are plotted as Required Air Pressure 10 bar (145psi) linear piston displacement versus Intensifier Displacement 3.6 mL pressure in thousands of psi. Since diameter of the piston (9.525 mm) remains constant, linear displacement in Test Setup a leak-free system is directly proportional to the volume change and, therefore, ΔV The HUB 880 intensifier, a tee with can be derived. The system records both a high pressure transducer, a pressure and volume change values section of high pressure tubing and automatically over a wide range of

the manual high pressure valve mm displacement, piston Linear pressure values. Resulting data contain (total maximal volume is 5.1 mL) Data for ten consecutive triangular sufficient information to calculate bulk was used as a sample container. waves are plotted for each sample modulus of the sample at given Intensifier piston displacement (full temperature. The HUB880 instrument is stroke) was 3.6 mL. capable of programmable temperature Degassed liquid samples were control, offering an ability of complex unattended compressibility tests. introduced via inlet check valve by a Pressure, psi x 103 % Disp syringe. System was purged of previous sample by an automatic 50 program containing 10 full intensifier strokes with pressurization to 1 kbar 45 HUB880 data bounded by between strokes to dissolve and 40 NIST constant temperature (25C) purge any remaining bubbles. and isentropic (.3kJ/kgK) data

35 High resolution linear encoder was mounted 30 directly onto an intensifier and coupled with a lever 25 connected to an intensifier 20 plunger between air and

water seals. Signal from 15 this piston position sensor was acquired by the 10 : Seattle Tap Water auxiliary ADC channel on the HUB880 control board. 5

0

0 10 20 30 40 50 60 70 80 90

HUB880 data appear superimposed onto the isothermal and isentropic water compressibility curves. Conclusions The NIST water compressibility data were used as a normalization standard for other sample types. Compressibility data for various are currently incomplete and only available for Sample selected temperature values [5-7]. Instrument described here offers an ability to acquire such data in automated fashion by manipulation of pressure and temperature and recording corresponding volumetric displacement. Correction for the seal friction can be derived form the acquired data and experimental values of bulk modulus can be Water 4.55 4.591 obtained. Our data suggests that the system provides enough resolution and accuracy to match published data, when available. The built-in software temperature control and Ethanol 11.28 11.19 optional resistive heating blanket can be used to automatically acquire compressibility date for a broad range of without reloading the sample. Better Methanol 14.81* 12.14 understanding of compressibility of aqueous solutions, organic solvents and their 2-Propanol 13.45 13.32 mixtures with water may provide useful information for development of improved HPP processes for juices and alcoholic beverages. Moreover, such data may help to further Olive Oil 5.87 n/a improve instrumentation and consumables for ultra high pressure liquid chromatography (UHPLC), as well as many more useful applications. * Possible evaporation insideCanola intensifier Oil during sample aspiration5.16 resulting in higher thann/a expected compressibility

References [1] Tomin A, Lazarev A, Bere MP, Redjeb H, Török B. Selective reduction of ketones using water as a hydrogen source under high hydrostatic pressure. Org. Biomol. Chem. 2012, 10, 7321-7326. [2] J. McCoy, W. L. Hubbell. High-pressure EPR reveals conformational equilibria and volumetric properties of spin-labeled proteins. Proc Natl Acad Sci USA. 2011, 108(4):1331-6. [3] Ando N., Barstow B. High Hydrostatic Pressure Effects on Proteins: Fluorescence Studies. In: Encyclopedia of Analytical Chemistry, Online ©2006–2012 John Wiley & Sons, Ltd. [4] Munte CE, Beck Erlach M, Kremer W, Koehler J, Kalbitzer HR. Distinct conformational states of the Alzheimer β-amyloid peptide can be detected by high-pressure NMR spectroscopy. Angew Chem Int Ed Engl. 2013; 52(34):8943-7. [5] Lide, D. R., and Kehiaian, H. V., CRC Handbook of Thermophysical and Thermochemical Data, CRC Press, Boca Raton, FL, 1994. [6] Le Neindre, B., Effets des Hautes et Tres Hautes Pressions, in Techniques de l’Ingenieur, Paris, 1991. [7] CRC Handbook of Chemistry and Physics, 95th Edition, 2014-2015, CRC Press, Internet version 2015

HPBB 2014 Poster Copyrights 2014 Pressure BioSciences, Inc. www.pressurebiosciences.com